Molecular Epidemiology of Penicillinase-Producing Neisseria gonorrhoeae Isolates and Their blaTEM-135 Gene Variant in Bangkok, Thailand, 2015-2017.

Penicillinase-producing Neisseria gonorrhoeae (PPNG) possessing blaTEM-135 is a serious public health threat. With only a single change in the amino acid sequence, blaTEM-135 could evolve into a TEM-type extended-spectrum beta-lactamase (ESBL), which hydrolyzes extended-spectrum cephalosporins, including ceftriaxone and cefixime. We investigated the molecular epidemiological characteristics, types of plasmids in PPNG isolates, and prevalence of PPNG clinical isolates producing TEM-135 beta-lactamases. N. gonorrhoeae multi-antigen sequence typing (NG-MAST) was used to determine the molecular epidemiological characteristics of 99 PPNG isolates collected from 2015 to 2017. A mismatch amplification mutation assay was used to examine the blaTEM-135 gene prevalence. Of the 89 identified NG-MAST sequence types, 65 (73.0%) were novel. Only 17.7% (43/243) of PPNG isolates belonged to 16 genogroups. The most frequent plasmid was African, followed by Rio/Toronto, and Asian. The blaTEM-135 allele was found in Rio/Toronto plasmids. The blaTEM-135 allele was present in 23.2% (23/99) of the PPNG isolates. PPNG isolates expressing TEM-135 beta-lactamase exhibited significantly higher penicillin MIC (minimum inhibitory concentration) values than TEM-1 PPNG isolates. The PPNG isolates showed high genetic diversity and a high proportion of blaTEM-135 alleles. Mutation of the blaTEM-135 allele is worrisome as only one mutation could cause TEM-1 to evolve into an ESBL variant that degrades ceftriaxone. Ongoing surveillance of blaTEM-135 and new PPNG isolates is imperative.

Penicillinase plasmid Australia type in Neisseria gonorrhoeae isolated in Poland.

PURPOSE: Neisseria gonorrhoeae is an etiological agent of gonorrhea which remains a major public health problem the mechanisms that determine resistance to drugs of the beta-lactam class, which are recommended for the treatment of gonorrhea, are currently the most important problem in its treatment. Chromosomal mutations are responsible for resistance to ceftriaxone and cefepime. The possibility of mutations in the gene encoding beta-lactamase (blaTEM) in the penicillinase plasmid may also turn out to be a serious threat. METHODS: The occurrence of resistance encoded on penicillinase plasmid has been investigated. For this purpose, the susceptibility of bacteria was determined and the gene for resistance to beta-lactams as well as the plasmids themselves was typed. RESULTS: Of the 333 strains tested, 21 (6.3%) had the beta-lactamase gene and produced penicillinase. Two of the beta-lactamase: TEM-1 and TEM-135 occurred among the tested strains of N. gonorrhoeae. Most of the known penicillinase plasmid types of N. gonorrhoeae were demonstrated: the Asian, the African, the Toronto/Rio plasmids and Australian variants. CONCLUSIONS: In the first 3 years, TEM-1 beta-lactamases dominated in N. gonorrhoeae, which were replaced by TEM-135 in the following years of the study. Not all molecular methods are capable of varying the types of penicillinase plasmids. A particularly noteworthy observation is the fact that the Australia-type of penicillinase plasmid (3270 bp) was identified for the first time in Europe, and the second time in the world.

Molecular characteristics and antimicrobial susceptibility of penicillinase-producing Neisseria gonorrhoeae isolates in Fukuoka, Japan, 1996-2018.

OBJECTIVES: The objective of this study was to investigate the molecular characteristics and antimicrobial susceptibility of penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates collected in Fukuoka, Japan, from 1996-2018. METHODS: Antimicrobial susceptibility to seven antibiotics was determined by the agar dilution method. Molecular characteristics were determined by Sanger sequencing of the blaTEM allele, plasmid typing and N. gonorrhoeae multiantigen sequence typing (NG-MAST). Furthermore, full sequences of the penA gene, encoding penicillin-binding protein 2 (PBP2), of PPNG isolates with reduced susceptibility to cefixime were analysed. RESULTS: Among 50 PPNG isolates, 17 and 33 were collected during 1996-2006 and 2007-2018, respectively. In 1996-2006, blaTEM-1 in African plasmid was most frequent (64.7%), followed by blaTEM-1 in Asian plasmid (29.4%) and blaTEM-135 in Toronto/Rio plasmid (5.9%). In 2007-2018, blaTEM-135 in Toronto/Rio plasmid was predominant (54.5%), followed by blaTEM-1 in African plasmid (36.4%) and blaTEM-135 in Asian plasmid (6.1%). Among isolates with the blaTEM-135-carrying Toronto/Rio plasmid in 2007-2018, a novel genogroup G15576 was predominant (66.7%). Isolates with the TEM-135 ß-lactamase were more resistant to ciprofloxacin but were more susceptible to ceftriaxone and tetracycline than isolates with TEM-1. Seven PPNG isolates less susceptible to cefixime possessed the plasmidic blaTEM-1 allele and had mosaic or non-mosaic alterations within PBP2. CONCLUSION: The proportion of PPNG with the blaTEM135-carrying Toronto/Rio plasmid increased during the last 12 years. The increase in PPNG carrying the blaTEM-135 allele is of particular concern as it is considered a possible direct precursor of an extended-spectrum ß-lactamase (ESBL).

Molecular Characteristics of "BlaB-Like" Chromosomal Inducible Cephalosporinase of Yersinia enterocolitica Biotype 1A Strains.

Yersinia enterocolitica biotype 1A strains are emerging pathogens, frequently isolated from clinical samples across the globe. Y. enterocolitica strains produce two chromosomal ß-lactamases, BlaA and BlaB. BlaA is a constitutively expressed, Ambler class A, penicillinase, whereas BlaB is Ambler class C-type, inducible cephalosporinase. An earlier study from our laboratory indicated that instead of BlaB, Y. enterocolitica biotype 1A produced a "BlaB-like" enzyme. The objective of this work was to study the molecular characteristics of "Bla-B like" ß-lactamases produced by biotype 1A strains to discern their similarity with AmpC-type ß-lactamases and the basis of varied levels of minimum inhibitory concentration (MIC) to cefotaxime. Thus, the promoters and blaB genes were investigated in four strains of biotype 1A. Three-dimensional structures of the "BlaB-like" enzymes were modeled, and docked in silico with cefotaxime to understand how specific substitutions in gene sequences affect antibiotic susceptibilities. Our results indicated that all the reported key catalytic residues were present in variants of "Bla-B-like" enzymes, discerned in biotype 1A strains, but at different positions. Molecular docking of enzyme variants with cefotaxime revealed that lesser was the number of the H-binding residues with cefotaxime in a strain, lower was the MIC of cefotaxime in that strain. To the best of our knowledge, this is the first study in which the molecular characteristics and enzymatic interactions of "BlaB-like" cephalosporinases of Y. enterocolitica biotype 1A strains have been reported.

Molecular Characterization of Penicillinase-Producing Neisseria gonorrhoeae Isolated in Two Time Periods, 2003-2004 and 2014-2015, in Italy.

The emergence of antibiotic resistant strains poses a great concern for gonorrhea treatment. The aim of this study was to characterize penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates collected in Italy in two time frames, 2003-2004 and 2014-2015. A total of 80 PPNG were characterized for the blaTEM gene variant and the plasmid type. Furthermore, gonococci were typed using Neisseria gonorrhoeae multiantigen sequence typing. Antibiotic susceptibility assay was performed for penicillin, ciprofloxacin, ceftriaxone, and spectinomycin by Etest and minimum inhibitory concentration (MIC) test strip methods. The ß-lactamase production was detected using nitrocefin test. Among PPNG isolates, four blaTEM alleles were identified as follows: blaTEM-1, blaTEM-228, blaTEMP14S, and blaTEM-135. The African plasmid possessed the blaTEM-1, blaTEM-228, and blaTEMP14S, whereas blaTEM-135 was identified in Toronto/Rio and Asian plasmids. The percentage of isolates with the blaTEM-1-carrying African plasmid increased from 42.5% in 2003-2004 to 55% in 2014-2015; conversely, the isolates with blaTEM-135-carrying Toronto/Rio plasmid decreased from 57.5% to 35%. Among the isolates carrying the Toronto/Rio plasmids possessing blaTEM-135, sequence type (ST)661 and ST5624 were found to be the predominant STs in both periods 2003-2004 and 2014-2015, respectively. More than half of the PPNG isolates were resistant to ciprofloxacin. Increase in the isolates carrying the African plasmid possessing blaTEM-1 and a parallel decrease of the blaTEM-135-carrying Toronto/Rio plasmid was observed. Moreover, PPNG isolate harbored Toronto/Rio plasmid with blaTEM-135 belonged mainly to two major STs (ST661 and ST5624). Given the possible role of a mutated blaTEM gene as an additional mechanism to extended spectrum ß-lactamase resistance, it is crucial to monitor gonococci carrying these resistance genes.

Prevalence of blaZ Gene and Performance of Phenotypic Tests to Detect Penicillinase in Staphylococcus aureus Isolates from Japan.

Guidelines recommend that clinical laboratories perform phenotypic tests (nitrocefin-based test and penicillin 10-U [P10] or 1-U [P1] zone edge tests) to detect penicillinase in Staphylococcus aureus isolates. This study aimed to assess the prevalence of blaZ encoding penicillinase and perform various phenotypic tests in S. aureus isolates from Japan. We prospectively collected 200 methicillin-susceptible S. aureus isolates from June 2015 to January 2016 and performed six phenotypic tests (nitrocefin-based test, P10 zone edge test/P10 diffusion test, penicillin 2-U [P2] zone edge test/P2 diffusion test, and cloverleaf test) on each sample. We confirmed the presence of blaZ (two blaZ-positive isolates) using PCR. Using blaZ PCR as a standard, we observed a low sensitivity (50%) and positive predictive value (PPV, 50%) of the nitrocefin-based test, low PPV (18.2%) of the P10 zone edge test, low sensitivity (50%) of the P10 diffusion test, low PPV (50% and 22.2%) of the P2 zone edge test and P2 diffusion test, respectively, and low sensitivity (50%) of the cloverleaf test. These data suggest a low performance (sensitivity and PPV) of these six phenotypic tests because of the low prevalence (1%) of blaZ in S. aureus isolates from Japan.

A combination of mecillinam and amoxicillin/clavulanate can restore susceptibility of high-level TEM-1-producing Escherichia coli to mecillinam.

Objectives: Mecillinam is recommended in France as a first-line treatment for lower urinary tract infections, due to the large increase in resistance of Escherichia coli to other oral treatments, such as co-trimoxazole or fluoroquinolones, its limited impact on faecal microbiota and its stability in the presence of numerous ß-lactamases. However, we recently identified several mecillinam-resistant E. coli isolates with a high-level expression penicillinase (HEP) phenotype that merit further study. Patients and methods: We studied two isogenic clinical isolates from one patient (one susceptible to mecillinam and one resistant to mecillinam) by WGS to determine the mechanism of mecillinam resistance and compared it with other mecillinam-resistant E. coli . We evaluated the synergistic combination of amoxicillin/clavulanate and mecillinam using a simple test, suitable for daily laboratory practice, to determine the MIC of this combination. Results: We showed that the presence of an SNP in the promoter of the plasmidic TEM-1 ß-lactamase gene is sufficient to confer resistance to mecillinam. This mechanism was present in 67% of HEP-phenotype E. coli tested. Combining mecillinam with amoxicillin/clavulanate abolished resistance, with an MIC compatible with clinical use. This association was not sensitive to the inoculum effect, in contrast to mecillinam alone. Conclusions: An HEP phenotype can confer mecillinam resistance in vitro . This resistance is abolished, regardless of the inoculum, by combining mecillinam with amoxicillin/clavulanate, and can be easily tested in the laboratory. This combination may be used as an oral relay treatment of non-complicated pyelonephritis due to multiresistant E. coli strains.

Molecular epidemiology of penicillinase-producing Neisseria gonorrhoeae isolates in France.

OBJECTIVES: Characterizing the molecular epidemiology of antibiotic resistance is crucial for a better understanding of the evolution and spread of resistance in Neisseria gonorrhoeae. Here, we examine the molecular epidemiology of penicillinase-producing N. gonorrhoeae (PPNG) isolates in France. METHODS: We investigated 176 PPNG isolates collected between 2010 and 2012 by the National Reference Centre in France. Genotyping was performed using the NG-MAST technique, blaTEM genes were Sanger-sequenced, and plasmids were characterized by PCR-typing. RESULTS: We revealed the existence of four major clusters representing about one-third of PPNG circulating in France. These clusters were related to ST1479 (18/176, 10.2%), to ST1582 (15/176, 8.5%), to ST8922 (10/176, 5.6%), and to ST1285 (9/176, 5.1%). Wild-type TEM-1 was identified in 151 (151/176, 85.8%) PPNG isolates, and TEM-1 variants were mostly represented by the M182T mutation (14/176, 8%), followed by P14S/L (8/176, 4.5%), G228S (2/176, 1.1%), and Q269K (1/176, 0.6%). The blaTEM genes were carried by African (157/176, 89.2%), Asian (13/176, 7.4%), and Toronto/Rio (6/176, 3.4%) plasmids. The M182T variants were found in various genetic backgrounds, whereas the P14S variants were disseminated clonally. The G228S and Q269K variants belong to one of the four major clusters of PPNG, which suggests a recent de novo emergence of these mutations. CONCLUSIONS: Our results show that approximately one-third of the penicillinase-producing N. gonorrhoeae isolates in France belong to one of four major clusters and that the spread of the different TEM variants is associated with distinct patterns of molecular epidemiology.

Chromosome-Encoded Broad-Spectrum Ambler Class A ß-Lactamase RUB-1 from Serratia rubidaea.

Whole-genome sequencing of Serratia rubidaea CIP 103234T revealed a chromosomally located Ambler class A ß-lactamase gene. The gene was cloned, and the ß-lactamase, RUB-1, was characterized. RUB-1 displayed 74% and 73% amino acid sequence identity with the GIL-1 and TEM-1 penicillinases, respectively, and its substrate profile was similar to that of the latter ß-lactamases. Analysis by 5' rapid amplification of cDNA ends revealed promoter sequences highly divergent from the Escherichia coli σ70 consensus sequence. This work further illustrates the heterogeneity of ß-lactamases among Serratia spp.

Efficient plasmid transduction to Staphylococcus aureus strains insensitive to the lytic action of transducing phage.

The transduction mediated by bacteriophages is considered to be one of the primary driving forces in horizontal gene transfer in staphylococci, which is crucial to their adaptation and successful evolution. For a transduction to be effective, it is generally accepted that the recipient strain should be susceptible to the transducing phage. In this study, we demonstrate that the plasmid DNAs are effectively transduced into the recipient Staphylococcus aureus strains in spite of their insensitivity to the lytic action of the transducing phage, provided that these phages adsorb effectively to the bacterial cells. The tetracycline and penicillinase plasmids were transduced to insensitive laboratory and clinical strains by bacteriophages ϕ29, ϕ52A and ϕ80α as well as by prophage ϕ53 and naturally occurring prophages induced from donor lysogenic strains. Comparable frequencies of transduction were achieved in both phage-sensitive and phage-insensitive recipient strains. We have demonstrated that such mechanisms as the restriction of DNA and lysogenic immunity which are responsible for insensitivity of cells to phages may not be a barrier to the transfer, maintenance and effective spread of plasmids to a wider range of potential recipients in the staphylococcal population.

A penicillin- and metronidazole-resistant Clostridium botulinum strain responsible for an infant botulism case.

The clinical course of a case of infant botulism was characterized by several relapses despite therapy with amoxicillin and metronidazole. Botulism was confirmed by identification of botulinum toxin and Clostridium botulinum in stools. A C. botulinum A2 strain resistant to penicillins and with heterogeneous resistance to metronidazole was isolated from stool samples up to 110 days after onset. Antibiotic susceptibility was tested by disc agar diffusion and MICs were determined by Etest. Whole genome sequencing allowed detection of a gene cluster composed of blaCBP for a novel penicillinase, blaI for a regulator, and blaR1 for a membrane-bound penicillin receptor in the chromosome of the C. botulinum isolate. The purified recombinant penicillinase was assayed. Resistance to ß-lactams was in agreement with the kinetic parameters of the enzyme. In addition, the ß-lactamase gene cluster was found in three C. botulinum genomes in databanks and in two of 62 genomes of our collection, all the strains belonging to group I C. botulinum. This is the first report of a C. botulinum isolate resistant to penicillins. This stresses the importance of antibiotic susceptibility testing for adequate therapy of botulism.

Dominating types of penicillinase-plasmids in Neisseria gonorrhoeae strains isolated in 2010-2012 in Warsaw.

INTRODUCTION: The reason of Neisseria gonorrhoeae resistance to penicillin is often production of TEM beta-lactamases encoded by plasmids. The most common types of the plasmid are Africa, Asia and Toronto/Rio. Another reason of resistance can be mutations in bacterial chromosome. The aim of the study was to investigate the types of plasmids occurring in in Neisseria gonorrhoeae strains isolated in 2010-2012 in Warsaw. MATERIAL AND METHODS: From 218 isolated in 2010, 2011 and at the beginning of 2012 from patients of Medical University in Warsaw we selected 12 strains producing beta- lactamase (penicillinase producing N. gonorrhoeae, PPNG). d B-tests to investigate bacterial sensitivity to penicillin and cefiriaxon. The types of plasmids were determined with PCR. RESULTS: The Beta-lactamases were encoded by Toronto/Rio (41,7%), Asia (33,3%) and Africa (25,0%) plasmids. All the strains were resistant to penicillin (MIC 2-8 mg/L) and sensitive to ceftriaxon (MIC 0,004-0,032 mg/L). CONCLUSIONS: All of the investigate PPNG strains were penicillin resistant and ceftriaxon sensitive. The dominating type of the penicillinase plasmid was Toronto/Rio.

The prevalence and epidemiology of plasmid-mediated penicillin and tetracycline resistance among Neisseria gonorrhoeae isolates in Guangzhou, China, 2002-2012.

BACKGROUND: Gonococcal antimicrobial resistance is a global problem. Different resistance plasmids have emerged and spread among the isolates of Neisseria gonorrhoeae worldwide and in China. We conducted this study to monitor the plasmid-mediated penicillin and tetracycline resistance among N. gonorrhoeae isolates in Guangzhou from 2002 to 2012. METHODS: Consecutive isolates of N. gonorrhoeae were collected from outpatients with gonorrhea attending the STD clinic in Guangdong Provincial Centre for Skin Diseases and STIs Control and Prevention. Penicillinase-producing N. gonorrhoeae (PPNG) isolates were analyzed by the paper acidometric method. Plasmid-mediated resistance to tetracycline in N. gonorrhoeae (TRNG) isolates was screened by the agar plate dilution method. Plasmid types were determined for TRNG and PPNG isolates using polymerase chain reaction (PCR). Minimum inhibitory concentrations (MICs) to penicillin and tetracycline were detected by the agar plate dilution. RESULTS: Of 1378 consecutive N. gonorrhoeae isolates, 429 PPNG and 639 TRNG isolates were identified. The prevalence of PPNG, TRNG, and PPNG/TRNG increased from 18.3 to 47.1 % (χ (2) = 31.57, p < 0.001), from 29.4 to 52.1 % (χ (2) = 16.28, p < 0.001) and from 10.0 to 26.2 % (χ (2) = 10.46, p < 0.001) between 2002 and 2012, respectively. Genotyping of plasmids among PPNGs showed that the majority (93.7 %) of the isolates were the Asian type plasmids, while the African type plasmid emerged in 2008 and rapidly increased to 14.0 % in 2012 (χ (2) = 25.03, p < 0.001). For TRNGs, all 639 isolates carried the Dutch type plasmid. MICs of penicillin G and tetracycline persisted at high levels and the MIC90s were 32-fold higher than the resistant cutoff point over 11 years. The prevalence rates of penicillin- and tetracycline-resistant N. gonorrhoeae varied from 90.9 to 91.1 % and from 88.3 to 89.3 % during 2002 to 2012, respectively. CONCLUSIONS: Resistance to penicillin and tetracycline among N. gonorrhoeae isolates remained at high levels in Guangzhou. The Asian type PPNG continued to spread and Dutch type TRNG was still the dominant strain. The African type PPNG has emerged and is spreading rapidly.

Genetic diversity of blaTEM alleles, antimicrobial susceptibility and molecular epidemiological characteristics of penicillinase-producing Neisseria gonorrhoeae from England and Wales.

OBJECTIVES: The objective of this study was to investigate the genetic diversity of blaTEM alleles, antimicrobial susceptibility and molecular epidemiological characteristics of penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates collected in 2012 from England and Wales. METHODS: PPNG isolates were from the 2012 Gonococcal Resistance to Antimicrobial Surveillance Programme (GRASP). Their susceptibility to seven antimicrobials was determined using agar dilution methodology. ß-Lactamase production was detected using a nitrocefin test. ß-Lactamase plasmid types were determined and blaTEM genes were sequenced. Isolates were also typed by N. gonorrhoeae multi-antigen sequence typing (NG-MAST). RESULTS: Seventy-three PPNG isolates were identified in the 2012 GRASP collection (4.6%, 73/1603). Three different blaTEM alleles were identified, encoding three TEM amino acid sequences: TEM-1 (53%), TEM-1 with a P14S substitution (19%) and TEM-135 (27%). The blaTEM-135 allele was present in nine different NG-MAST types and was found mostly on Asian (60%) and Toronto/Rio (35%) plasmids. By contrast, most TEM-1-encoding plasmids were African (98%). All the TEM-135 isolates displayed high-level ciprofloxacin and tetracycline resistance. CONCLUSIONS: The high proportion of blaTEM-135 alleles (27%) demonstrates that this variant is circulating within several gonococcal lineages. Only a single specific mutation near the ß-lactamase active site could result in TEM-135 evolving into an ESBL. This is concerning particularly because the TEM-135 isolates were associated with high-level ciprofloxacin and tetracycline resistance. It is encouraging that no further TEM alleles were detected in this gonococcal population; however, vigilance is vital as an ESBL in N. gonorrhoeae would render the last remaining option for monotherapy, ceftriaxone, useless.

Multitarget PCR Assay for Direct Detection of Penicillinase-Producing Neisseria gonorrhoeae for Enhanced Surveillance of Gonococcal Antimicrobial Resistance.

A multitarget PCR was developed for the direct detection of penicillinase-producing Neisseria gonorrhoeae (PPNG). The assay was validated by testing 342 PPNG isolates and 415 clinical samples. The method is suitable for routine detection of PPNG strains. Its multitarget approach reduces the potential for false-negative results caused by sequence variations.

Penicillinase-producing plasmid types in Neisseria gonorrhoeae clinical isolates from Australia.

Penicillinase-producing Neisseria gonorrhoeae (PPNG) carrying the blaTEM-135 gene is of particular concern, as it is considered a stepping stone toward resistance to extended-spectrum cephalosporins. Here, we sought to characterize plasmid types and the occurrence of the blaTEM-135 gene for N. gonorrhoeae clinical isolates from Australia. We found that blaTEM-135 was prevalent in Australian PPNG and was detected on all three major plasmid types.

First identification of a chromosomally located penicillinase gene in Kingella kingae species isolated in continental Europe.

Kingella kingae is the major pathogen causing osteoarticular infections (OAI) in young children in numerous countries. Plasmid-borne TEM-1 penicillinase production has been sporadically detected in a few countries but not in continental Europe, despite a high prevalence of K. kingae infections. We describe here for the first time a K. kingae ß-lactamase-producing strain in continental Europe and demonstrate the novel chromosomal location of the blaTEM-1 gene in K. kingae species.

Non-culture Neisseria gonorrhoeae molecular penicillinase production surveillance demonstrates the long-term success of empirical dual therapy and informs gonorrhoea management guidelines in a highly endemic setting.

OBJECTIVES: Unlike most of the world, penicillin resistance in Neisseria gonorrhoeae from remote regions of Western Australia (WA) with high gonorrhoea notification rates has not increased despite many years of empirical oral therapy. With the advent of non-culture molecular diagnosis of gonorrhoea and the consequent decline in culture-based susceptibility, it is imperative to ensure the ongoing reliability of combination oral azithromycin, amoxicillin and probenecid for uncomplicated gonorrhoea in this setting. PCR-based non-culture N. gonorrhoeae antimicrobial resistance surveillance for penicillinase production was therefore employed. METHODS: Genital and non-genital specimens that were PCR-positive for N. gonorrhoeae were assessed for penicillinase production by detection of the N. gonorrhoeae TEM-1 plasmid using specific real-time PCR. RESULTS: In remote regions of WA where gonorrhoea is highly endemic, <5% of N. gonorrhoeae isolates were penicillinase-producing. This contrasts with rates of up to 20% observed in the more densely populated metropolitan and rural regions. CONCLUSIONS: In the era of molecular diagnosis of gonorrhoea, non-culture-based antimicrobial resistance surveillance proved useful when developing evidence-based guidelines for the clinical management of locally acquired gonorrhoea in highly endemic regions in WA. The continued efficacy of combination oral amoxicillin, probenecid and azithromycin therapy despite many years of use in a setting highly endemic for gonorrhoea may explain the low rate of penicillin resistance in these remote regions and supports the concept of adding azithromycin to ß-lactam antibiotics to help delay the emergence of multiresistant N. gonorrhoeae.